LABS & PROTOCOLS | telhaiigem

30/06/2016

Aim of the lab :transformation of the E.coli bacteria

Tools and Methods: transformaton with heat shock and then growing the bacteria on LB Agar plates, with CM added antibiotics.

The Igem plasmid kit consisted of:

plasmid Psb1c3
plasmid BBa_k608008
plasmid BBa_J04450

Results: the transformation was succsecful (haser tmunut)

06/07/2016

Aim of the lab: extraction of the plasmid

Tools and Methods: gas collumn

Results:

13/07/2016

Aim of the lab:

Tools and Methods: restriction of pSB1C3 backbone and BBa_J04450 with EcoRI and PstI

We prepared 1% Agarose gel, and fed the restricted products through the gel.

Results:

U.C uncut plasmid

7/13/16
Aim of the lab: cutting the plasmids, in order to ensure desired sizes.
Important notes:
1.         We will only use two of the three plasmids we have. The plasmid comprised of GFP is to be used in future experiments (attaching the plasmid to the protein). It does not recognize an animal promotor, and therefore we will need to use a GFP plasmid that has an animal promotor.
2.         The restriction sites on the backbone of the plasmid are very close to each other, and hence we must cut each enzyme, in order to make sure that both cut as they should.
3.         The restriction sites upon the RFP plasmid are not in conjugation, and therefore, we will cut each of the enzymes separately, and in addition, we will cut one additional time with two enzymes simultaneously. We expect to receive 2 fragments, one comprised of 1000 bp and the other of 2000 bp.

Tools and methods
Preparation of 1% agarose
1.         Prepare the gel platter
2.         Weigh 1 gram of agarose, and add it to the arlenmeir, containing 100 ml of TAE buffer.
3.         Melt in the microwave for approximately 2 minutes
4.         Add 5 microliters of EtBr
5.         Pour the mixture into the platter and wait for it to harden.
Cutting reactions to the prepared plasmids
Cutting tubes

•           Incubate the tubes in the incubator at °37 C for 90 minutes.
•           Add to each tube 4 µliter of colour.
•           Running the followings on the Agarose gel in this order:
a.         λH3 size mark
b.         3 µliter out of the pSB1C3 plasmid of which was not cut.
c.         All of the cutting reaction of the pSB1C3   - PstI.
d.         All of the cutting reaction of the pSB1C3   - EcoRI.
e.         3 µliter of the J04450 plasmid of which was not cut.
f.          All of the cutting reaction of the J04450   - PstI.
g.         All of the cutting reaction of the J04450   - EcoRI.
h.         All of the cutting reaction of the J04450   - EcoRI-Pstl.

Results:

03/08/2016

Aim of the lab: Cutting DNA fragments and the plasmid, and ligation of the two

Biological parts used:

Plasmid- pSB1C3

Part 2- Donor

Part 3- gRNA1

Part 4- gRNA2

For each of the fragments, we cut according to the following protocol:

Part 8 ul

PstI 0.5 ul

EcoRI 0.5ul

Bufx10 1ul

With regards to the plasmid, the cutting was done as follows:

pSB1C3 5 ul

PstI 1 ul

EcoRI 1 ul

Bufx10 2 ul

DDW 11 ul

The tubes were incubated for one hour at 37 degrees celcius.

The ligations were done as follows:

Lig1 – pSB1C3+ part2

Lig2 – pSB1C3+ part3

Lig3 – pSB1C3+ part4

Each tube containing the fragments undergoing ligation, contained the following mixture:

pSB1C3 4 ul

part 8.5 ul

T4 ligase 1 ul

Bufx10 1.5 ul

The tubes were incubated at 16 degrees celcius overnight, and during the following morning, transformation was made within E Coli TOP10 bacteria, and the bacteria was spread onto petri plates containing chloramphenicol.

07/08/2016

As a result of the ligations we performed, the following colonies grew on the petri plates:

Lig1 – pSB1C3+ part2

Lig3 – pSB1C3+ part4

From each system, 6 colonies were raised and transferred to a liquid medium containing chloramphenicol and were left to continue growing overnight.

Throughout the following morning, the plasmids were isolated and ran through agarose gel (1%).

After the plasmids were fed through the agarose gel, the following splicing reaction was performed:

Plasmid 4 ul

EcoRI 0.75 ul

PstI 0.75 ul

Bufx10 1.5 ul

DDW 8 ul

The tubes were stored for one hour at 37 degrees celcius and then were fed through agarose gel (1%).

We can see from the picture above that from pSB1C3+ part4 we received fragments containing 479 bp (relatively large), indicating that they contain the gRNA2 element within them.